research article phase separation behavior and system...

Research ArticlePhase Separation Behavior and System Properties ofAqueous Two-Phase Systems with Polyethylene Glycol andDifferent Salts Experiment and Correlation

Haihua Yuan1 Yang Liu1 Wanqian Wei1 and Yongjie Zhao2

1Department of Biology and Guangdong Provincial Key Laboratory of Marine Biotechnology Shantou University ShantouGuangdong 515063 China2Department of Mechanical Engineering College of Engineering Shantou University Shantou Guangdong 515063 China

Correspondence should be addressed to Yang Liu liuyanglftstueducn

Received 15 October 2014 Revised 22 January 2015 Accepted 3 February 2015

Academic Editor Robert M Kerr

Copyright copy 2015 Haihua Yuan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The phase separation behaviors of PEG1000sodium citrate PEG4000sodium citrate PEG1000ammonium sulfate andPEG4000ammonium sulfate aqueous two-phase systems were investigated respectively There are two distinct situations for thephase separation rate in the investigated aqueous two-phase systems one state is top-continuous phase with slow phase separationrate and strong bottom-continuous phase with fast phase separation rate and weak volume ratio dependenceThe system propertiessuch as density viscosity and interfacial tension between top and bottom phases which have effects on the phase separationrate of aqueous two-phase systems were measured The property parameter differences between the two phases increased withincreasing tie line length and then improved the phase separation rate Moreover a modified correlation equation including thephase separation rate tie line length and physical properties of the four aqueous two-phase systems has been proposed andsuccessfully tested in the bottom-continuous phase whose coefficients were estimated through regression analysis The predictedresults of PEG1000sodium citrate aqueous two-phase systemswere verified through the stationary phase retention in the cross-axiscountercurrent chromatography

1 Introduction

In the 1960s aqueous two-phase systems (ATPS) have beenexploited to process different biological sources for therecovery of biological products such as amino acids proteinsnucleic acids antibodies cells and organelles [1ndash5] ATPShave also become an attractive separation technology dueto the mild conditions the low cost of the phase formingmaterials the simplicity of the process and the easy scale-upto an industrial level Recently polymersalt ATPS especiallypolyethylene glycol (PEG)salt ATPS have been extensivelyapplied in the separation of many biological products [6ndash10]PEGsalt ATPS have advantages of low cost low viscosityof both the phases and rapid phase separation rate so thiskind of ATPS has been used for the production of biologicalmolecules on an industrial scale For example the scale-upisolation of formate dehydrogenase from 30ndash50Kg of wet

cells ofCandida boidiniiwas investigated by Kroner et al [11]The separation of engineered protein was also successfullyscaled up to 1200L by Selber et al [12] It displays thatATPS have potential for the large-scale implementation inbiotechnology

Multistage extraction process such as high-speed coun-tercurrent chromatography (HSCCC) has been applied fur-ther to improve the target product purity compared with thesingle-stage extraction Organicaqueous solutions systemhas been widely used in HSCCC due to the short phase sepa-ration time (lt30 s) [13] while ATPS application in HSCCC islimited due to the long phase separation time (gt50 s) Thephase separation time of ATPS increases to 3ndash5min with theincreasing components concentrations [14 15] Moreover theemulsion phenomenon in ATPS is usually serious due to thelong phase separation time in the continuous process whichmakes it difficult to separate the target products thoroughly

Hindawi Publishing CorporationJournal of FluidsVolume 2015 Article ID 682476 10 pageshttpdxdoiorg1011552015682476

2 Journal of Fluids

Many works focused on HSCCC instrument design forATPS separation to eliminate the emulsion [16ndash18] Thereare few reports about the phase separation rate and physicalproperties of ATPS [14 15] for HSCCC

Much of the research [19 20] in ATPS has found thatpolyethylene glycolorganic salts ATPS could be employedas a viable and potentially useful tool for separating proteinsinstead of the conventional PEGinorganic systemsThemainadvantages of PEGsodium citrate ATPS are the biodegrad-ability and nontoxicity of the organic anion when comparingwith the high eutrophication potential of inorganic ions [21]and its harmful impact on the environment In our presentwork the physical properties and phase separation behav-iors of the ATPS including PEGorganic salts (PEGsodiumcitrate) and PEGinorganic salts (PEGammonium sulfate)systemswere investigated for comparisons Here ammoniumsulfate was selected as the inorganic salt due to its frequentuse in protein precipitation The phase separation rate andthe physical properties of ATPS such as density viscosity andinterfacial tension were measured and correlated with the tieline length (TLL) and ATPS 119881

119903which can contribute to the

HSCCC application using ATPS for protein separationFinally the stationary phase retention of PEGsodium citrateATPS in the cross-axis countercurrent chromatography (119883-axis CCC) was investigated based on the above correlatedmodels in which the 119883-axis CCC was designed and fabri-cated in our laboratory

2 Materials and Methods

21 Chemicals PEG1000 and PEG4000 (GR ge 95 masspurity) were purchased from Merck (Shanghai China)Sodium citrate (GR ge 995 mass purity) and ammoniumsulfate (GR ge 995 mass purity) were of analytical gradefrom local sources Aqueous solutions were prepared withdeionized and doubly distilled water

22 Preparation of ATPS ATPS were prepared from stocksolutions of PEG (50ww) sodium citrate (30ww) andammonium sulfate (40ww) Phase boundaries of all ATPSwere obtained by cloud-point measurements in a thermo-static bath (DC-0506 Shanghai Jingtian Electronic Instru-ment Co Ltd) of 25∘CThe temperature was maintained withan uncertainty of plusmn005∘C The PEGsalt ATPS with five tielines lengths (30 35 40 45 and 50) were equilibrated for 12 hat 25 plusmn 005

∘C before use The compositions of the tie lineswere determined by the119881

119903and the lever rule [22]The119881

119903was

determined in graduated centrifuge tubeswith an uncertaintyof plusmn01mL

23 Density Measurements The top and bottom phase den-sities were measured using a 10mL density bottle with anuncertainty of plusmn01 kgsdotmminus3 at 25 plusmn 005

∘CThemeasurementswere done in triplicate and the average value was reported

24 Viscosity Measurements The viscosity of all ATPS wasmeasured using digital rotary viscosimeter (NDJ-5S Shang-hai Jingtian Electronic Instrument Co Ltd) with a precision

of plusmn00001 PasdotsThe samples were first maintained at workingtemperature in thermostatic bath with an uncertainty ofplusmn005∘C for 10min and measurements were done in tripli-cate

25 Interfacial TensionMeasurements The interfacial tensionwas measured by a drop volume method [23 24] at 25∘CSome burettes having capillaries with different outer diame-ters were used since the interfacial tension changed with thecomposition of ATPS Interfacial tension between the twophases was determined by the numbers and volumes of fallendrops from the top phase solution and the density differencebetween the top and bottom phasesThe interfacial tension isthen given by

120574 =

Δ120588119881119892

2120587119903120595

120595 = 09045 minus 07294 (

119903

11988113

) + 04293 (

119903

11988113

)

2

(03 lt

119903

11988113

lt 12)

120595 = 1007 minus 1479 (

119903

11988113

) + 1829 (

119903

11988113

)

2

(

119903

11988113

le 03)

(1)

where 120574 is the interfacial tension between the phases of ATPSΔ120588 is the density difference of the two phases119881 is the averagevolume of drops 119903 is the radius of the tip of burette 119892 isthe local value for the acceleration due to gravity and 120595 isthe correction factors determined by the values of 119881 and 119903The uncertainty of interfacial tension measurement wasplusmn00001mNsdotmminus1 All the measurements were done in trip-licate

26 Phase Separation Rate ATPS mixing was performed incentrifuge tubes with 10mL volume The time of mixing was10min bymanual operation For all the experiments the timefor phase separation was defined as the time needed for mostbulk of the phases to separate and a horizontal interface wasformed Some small drops of one phase which remained inthe other phase were ignored The measurements were donein triplicate with a precision of plusmn01 s

Kaul et al found that the kinetic behavior depends greatlyon which of the phases is continuous and that the propertiesof the continuous phase strongly influence the movement ofthe drops of the dispersed phase [25ndash27] Two typical batchesrsquoseparation is shown in Figure 1 For the top-continuousATPSthe drops of bottom phase descend in centrifuge tubes andform a settling front for the bottom-continuous ATPS thedrops of top phase ascend in centrifuge tubes and form arising front It can be seen that the process of coalescence isslower than droplet descent and ascent due to the existence ofthe front

The problems of phase continuity and phase inversionare important for the phase separation behavior and phase

Journal of Fluids 3

Figure 1 Two typical batch separations for PEG4000ammoniumsulfate ATPS Left top-continuous phase right bottom-continuousphase

separation rate It has been shown that the phase continuityand phase inversion occurred as the119881

119903changed according to

Kaul et alrsquos methods [28] During the ATPS phase separationprocess when the descending droplets in the dispersiveregionwere observed it means a top-continuous phase whileascending droplets means a bottom-continuous phase Forthe same TLL of ATPS different composition concentrationswith various119881

119903make different continuous phaseHence both

the top-continuous region and bottom-continuous regioncan be seen in the same phase diagram A locus of phaseinversion points is found in the phase diagram between top-continuous region and bottom-continuous region creatinga phase inversion band To determine this band four ATPSwith five different TLL were chosen and the separationbehavior of the ATPS at TLL with different 119881

119903was investi-

gatedAn equation can express the correlation between the

phase separation rates and the physical properties of PEGsaltATPS The comprehensive efficiency of the physical prop-erties including density viscosity and interface tension canbe represented by Morton number (119872

0) [29ndash31] Hence a

generalized correlation equation given by Mistry and Golob[4 27] was developed for the ATPS with different PEGmolarmasses and salt relating the Morton number (119872

0) the ratio

of interfacial tension to surface tension 119881119903 and TLL

119889ℎ

119889119905

(

120583119888

120582

) = 1198861198720

119887

(

120574

120590119888

)

119888

(

119881tp

119881bp)

119889

(TLL)119890 (2)

1198720=

Δ1205834

119892

1205743Δ120588

(3)

where 119886 119887 119888 119889 119890 are the coefficients of the equation 119889ℎ119889119905 isthe phase separation rate 120583

119888is the viscosity of the continuous

phase 120590119888is the surface tension of continuous phase119881tp is the

volume of top phase 119881bp is the volume of bottom phase andΔ120583 is the viscosity difference between top and bottom phases

27 Stationary Phase Retention in the Cross-Axis Countercur-rent Chromatography The cross-axis countercurrent chro-matography (119883-axis CCC) was designed and fabricated inour laboratory The 119883-axis CCC apparatus is displayed asFigure 2 which includes the six separation columns at thetotal column capacity of 71mL Each column was tightlycoiled by the polytetrafluoroethylene (PTFE) tube with20mm inner diameter In view of the best experimentalpartition efficiencies of ATPS in 119883-axis CCC [32] the mea-surement of stationary phase retention of ATPS in our119883-axisCCC was designed as follows the bottom phase was selectedas mobile phase119883-axis CCC revolution direction was coun-terclockwise the elutionmodewas fromhead to tail and frominward to outward and the flow rate was 05mLmin Thespecific operations for the stationary phase retention mea-surement were the same as Shinomiyarsquos methods [33]

3 Results and Discussion

31 The Effect of TLL on ATPS Density Viscosity and Inter-facial Tension The effect of TLL on the density can be seenin Figure 3 It has been shown that the densities of both topand bottom phases are increasing as the TLL becomes longerCompared with the influence of TLL on the density of thetop phase the variation in density of the bottom phase is verysignificantThis is because the influence of the composition ofsalt on the density is larger than the influence of the compo-sition of PEG Furthermore the bottom is rich in salt and thetop phase is rich in PEG As a result the density differencebetween the bottom phase and the top phase increases withthe increasing TLL Moreover it can be seen that the PEGmolecular weight has no obvious effect on the density of thePEG-rich phase of ATPS compared to Figures 3(a) and 3(b)or Figures 3(c) and 3(d)

The viscosities of the top and bottom phases as a functionof the TLL are shown in Figure 4 Figure 4 shows that the vis-cosity of the top phase increases slightly with the increasingTLL Furthermore there is a marked change in viscosity ofPEG4000 ATPS as TLL increasing compared with PEG1000ATPS It is because the viscosities of ATPS increase with theincreasing molar mass of PEG at the same PEG concen-tration Perumalsamy and Murugesan have measured theviscosity of PEG solutions at 25∘C and observed the sameresult [34]

Figure 5 shows that the interfacial tension increases withthe increasing TLL for all the ATPS Mishima et al used thedrop volume technique and observed the same trend [35]The compositionsrsquo concentrations play a key role in the solu-tionrsquos interfacial tension In ATPS the compositionsrsquo concen-trations increase with the increase of TLL in both the topphase and the bottom phase Thus the interfacial tensions invarious ATPS are significantly enhanced with the increasingTLL

32 The Separation Behavior of ATPS The separation behav-iors of four ATPS are shown in Figure 6 As can be seen inFigure 6 to the left of the solid points the top phase of ATPSis continuous To the right of the hollow points the bottom

4 Journal of Fluids

(a) (b)

Figure 2 Photographs of our cross-axis countercurrent chromatography without the cover case (a) and the overall appearance (b)

25 30 35 40 45 50 55

108

112

116

120

124

Den

sity

(gm

L)

TLL (ww)

(a)

114

117

111

108

105

Den

sity

(gm

L)

25 30 35 40 45 50 55

TLL (ww)

(b)

108

112

116

120

124

128

Den

sity

(gm

L)

25 30 35 40 45 50 55

TLL (ww)

TopBottom

(c)

108

104

112

116

120

124

Den

sity

(gm

L)

25 30 35 40 45 50 55

TLL (ww)

TopBottom

(d)

Figure 3 Densities of the top phase and the bottom phase as a function of TLL at 25∘C (a) PEG1000ammonium sulfate ATPS (b)PEG4000ammonium sulfate ATPS (c) PEG1000sodium citrate ATPS and (d) PEG4000sodium citrate ATPS The error bars representthe standard deviations in triplicate experiments

Journal of Fluids 5

TLL (ww)28 32 36 40 44 48 52

0

10

20

30

40120578middot103

(Pamiddot

s)

(a)

0

80

20

40

60

120578middot103

(Pamiddot

s)

TLL (ww)28 32 36 40 44 48 52

(b)

TLL (ww)28 32 36 40 44 48 52

0

6

18

12

24

120578middot103

(Pamiddot

s)

TopBottom

(c)

TLL (ww)28 32 36 40 44 48 52

TopBottom

0

80

100

20

40

60120578middot103

(Pamiddot

s)

(d)

Figure 4 Viscosities of the top phase and the bottom phase as a function of TLL at 25∘C (a) PEG1000ammonium sulfate ATPS (b)PEG4000ammonium sulfate ATPS (c) PEG1000sodium citrate ATPS and (d) PEG4000sodium citrate ATPS The error bars representthe standard deviations in triplicate experiments

phase of ATPS is continuousThe regionwhich is called phaseinversion band between the solid points and hollow points onthe same TLL is a range of ambiguity Both top-continuousphase and bottom-continuous phase can be seen in thisregion of ATPS Within this region the continuity of thephase is affected not only by composition of the mixture butalso by the fluid dynamics The region of ambiguity becomeslarge with the increasing TLL and locates nearly at theconstant salt concentration line In Figure 6 the region ofbottom-continuous phase is almost 4 times larger than theregion of top-continuous phase Furthermore the middlepoints of TLL locate in the bottom-continuous phase Thisindicates that the four investigated ATPS of 119881

119903= 1 were

operated in the region of bottom-continuous phaseFigure 7 shows the profile of the phase separation time

changes with the increasing top phase ratio (119881119905= 1(119881

119903+ 1))

Phase inversion takes place near the point of 119881119905= 08 (119881

119903=

4) where the continuous phase can change into the dispersed

phase and a sudden change of phase separation behavior isobserved The bottom phase is continuous when the 119881

119905is

less than 08 and short phase separation times are observedwith the smooth variation The top phase is continuouswhen the 119881

119905is more than 08 and the dramatic increase of

phase separation time with the increasing 119881119905is observed It

indicates that ATPSwould be better to operate in the bottom-continuous region rather than top-continuous region

33 Correlation Equations of Phase Separation Since theATPS of 119881

119903= 1 have been widely applied in the separation of

biological products the phase separation rate of ATPS of119881119903=

1 has been investigatedThe surface tension of water (120590119908) can

be used in the equation rather than the surface tension ofcontinuous phase (120590

119888) Hence (2) can be modified as

119889ℎ

119889119905

(

120583119888

120582

) = 1198861198720

119887

(

120574

120590119908

)

119888

(TLL)119889 (4)

6 Journal of Fluids

TLL (ww)28 34 40 46 52

060

080

100

120

140

Inte

rfaci

al te

nsio

n (m

Nm

)

(a)

TLL (ww)28 34 40 46 52

000

020

040

060

080

100

Inte

rfaci

al te

nsio

n (m

Nm

)

(b)

TLL (ww)28 34 40 46 52

000

010

020

030

040

050

Inte

rfaci

al te

nsio

n (m

Nm

)

(c)

TLL (ww)28 34 40 46 52

000

020

040

060

080

Inte

rfaci

al te

nsio

n (m

Nm

)

(d)

Figure 5 Interfacial tension between the top phase and the bottom phase as a function of TLL at 25∘C (a) PEG1000ammonium sulfateATPS (b) PEG4000ammonium sulfate ATPS (c) PEG1000sodium citrate ATPS and (d) PEG4000sodium citrate ATPS The error barsrepresent the standard deviations in triplicate experiments

The value of the coefficients (119886 119887 119888 119889) can be calculated withthe experimental values (Tables 1 and 2) This was performedby nonlinear regression using 1stOpt 15 software (China)Using these values for the bottom-continuous region (4) canbe written as (5) and (6)

For the bottom-continuous region of PEGammoniumsulfate ATPS the correlation equation can be expressed

119889ℎ

119889119905

(

120583119887

120574

) = 358 times 10minus3

1198720

00080

(

120574

120590119908

)

minus18688

(TLL)07650

(5)

For the bottom-continuous region of PEGsodium citrateATPS the correlation equation can be expressed

119889ℎ

119889119905

(

120583119887

120574


1198720

01735

(

120574

120590119908

)

minus15403

(TLL)25750

(6)

The 1198772 (correlation index) of the two equations is 0960 for

PEGammonium sulfate ATPS and 0971 for PEGsodiumcitrate ATPS respectively Equation (5) was used to predict

the phase separation rate of the bottom-continuous regionof PEGammonium sulfate ATPS of 119881

119903= 1 with an average

absolute relative deviation (AARD) value of 705 Equation(6) was used to predict the phase separation rate of thebottom-continuous region of PEGsodium citrate ATPS of119881119903= 1 with an AARD value of 681 The values of the coef-

ficient indicate the difference of the phase separation ratesbetween the two ATPSThe two equations show that the TLLhas a greater effect on the PEGsodium citrate ATPS than thePEGammonium sulfate ATPSThe119872

0has a similar effect on

the phase separation rates of both ATPSBoth (5) and (6) have predicted the phase separation rates

of ATPS with 119881119903

= 1 For the batch separation the resultsof prediction can provide a reference since the stable ATPSwith 119881

119903= 1 are widely used For the continuous separation

such as HSCCC the phase separation rate is the mostimportant factor for the choice of solvent systems [36]Hence it is necessary to investigate the relationship betweenphase separation rate and physical properties of ATPS In thispaper as phase diagrams show in Figure 6 the bottom-continuous regions of the four investigated ATPS are fit forcontinuous separation due to the fast phase separation ratesand weak 119881

119903dependence

Journal of Fluids 7

0

0

01

01

02

02

03

03

04

04

05

Ammonium sulfate (ww)

PEG1000

(w

w)

(a)

0

01

02

03

04

05

06

0 01 02 03


PEG4000

(w

w)

(b)

0 01 02 03 04

Sodium citrate (ww)

0

01

02

03

04

05

PEG1000

(w

w)

(c)

0 01 02 03

Sodium citrate (ww)

000

010

020

030

040

050

PEG4000

(w

w)

(d)

Figure 6 Experimental equilibrium data obtained for the ATPS at 25∘C phase inversion band between the points at the same TLL (a)PEG1000ammonium sulfate ATPS (b) PEG4000ammonium sulfate ATPS (c) PEG1000sodium citrate ATPS and (d) PEG4000sodiumcitrate ATPS

000 020 040 060 080 100

0

200

400

600

800

Top phase ratio (vv)

Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(a)

0

100

200

300

400

500

000 020 040 060 080 100


Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(b)

Figure 7 Effect of the change in percentage top phase (119881119905

= 1(119881119903

+ 1)) on phase separation time for the ATPS at 25∘C (a)PEG4000ammonium sulfate ATPS and (b) PEG4000sodium citrate ATPS The error bars represent the standard deviations in triplicateexperiments

8 Journal of Fluids

Table 1 Experimental properties parameters ofPEG1000ammonium sulfate ATPS and PEG4000ammoniumsulfate ATPS at 25∘C

TLL Δ119905 120590119908

120574 Δ120583 120583119887

[ww] [s] [mNm] [mNm] [mPasdots] [mPasdots]

PEG1000ammoniumsulfate

30 9038 7197 0739 22130 212035 9346 7197 0846 24158 219340 9752 7197 0965 27053 214845 9644 7197 1140 28595 220550 10104 7197 1161 32305 2295


30 28062 7197 0272 25010 199035 24702 7197 0376 33085 186540 21888 7197 0439 41625 177545 18800 7197 0592 54005 189550 16410 7197 0805 67790 2210

Table 2 Experimental properties parameters of PEG1000sodiumcitrate ATPS and PEG4000sodium citrate ATPS at 25∘C

TLL Δ119905 120590119908

120574 Δ120583 120583119887


PEG1000sodiumcitrate

30 15618 7197 0083 5650 365035 13348 7197 0169 7780 372040 12202 7197 0249 9500 405045 11198 7197 0297 1168 442050 11132 7197 0405 14365 5235


30 27993 7197 0167 23165 243535 24785 7197 0305 31185 271540 24429 7197 0433 42110 294045 26705 7197 0579 55145 335550 26648 7197 0755 71985 4015

34 Stationary Phase Retention of PEG1000Sodium CitrateATPS in the 119883-Axis CCC PEG1000sodium citrate ATPSwere selected to investigate the positive effect of fast phaseseparation rate on the stationary phase retention in the cross-axis countercurrent chromatography According to the abovecorrelation equation (6) the shorter TLL of PEG1000sodiumcitrate ATPS could obtain the faster phase separation rate ofthe two phases Thus PEG1000sodium citrate ATPS withTLL = 30 (1654ww PEG1000 and 1396ww sodiumcitrate) was determined as the investigatedATPS for themea-surement of the stationary phase retention in our119883-axis CCCat various rotation rates Figure 8 displays that the effect ofthe 119883-axis CCC revolution speed on the stationary phaseretention of PEG1000sodium citrate ATPS with TLL = 30 Itcan be seen that the maximum value of stationary phaseretention was 5483 at 800 rpm which was slightly higherthan the reported stationary phase retention of ATPS in other119883-axis CCCs [32] It indicated that the phase separation rateof ATPS played an important role in the stationary phaseretention in HSCCC

3873

4438

4923

54835144

5041

30

35

40

45

50

55

60

400 500 600 700 800 900 1000 1100

Stat

iona

ry p

hase

rete

ntio

n (

)

Revolution speed (rpm)

Figure 8 Effect of revolution speed on stationary phase retentionof PEG1000sodium citrate in the 119883-axis CCC The error barsrepresent the standard deviations in triplicate experiments

4 Conclusion

The physical properties of PEGammonium sulfate andPEGsodium citrate ATPS had been studied The density ofbottom phase is larger than that of top phase and increaseswith increasing TLL The viscosity of the top phase is 3ndash35times larger than the bottom phase and increases with theincreasing TLL The interfacial tension between the phasesincreases with the increasing TLL

The bottom phase is continuous at high 119881119903value and

fast separation rate has been observed as well as the smoothvariationThe top phase is continuous at low119881

119903value and the

dramatic increase of phase separation time with the increas-ing 119881119903value was observed There is a phase inversion band

between the top-continuous region and bottom-continuousregionThephase inversion band is located at the constant saltconcentration line in the phase diagram Within this regionthe continuity of the phase is affected not only by the compo-sition of the ATPS but also by the fluid dynamics

The phase separation rate was correlated using amodifiedcorrelation equation and the coefficients were found Itshowed that the 119877

2 of the two equations were 0960 for PEGammonium sulfate ATPS and 0971 for PEGsodium citrateATPS respectively The correlation equations gave goodresults for the bottom-continuous ATPS with 119881

119903= 1 The

results may be useful for the choice of optimal conditions ofATPS nomatter in batch separation or continuous separationprocess The correlation equations were applied to estimatethe fast phase separation rate of PEG1000sodium citrateATPS and further to measure the stationary phase retentionof PEGsodium citrate ATPS in our119883-axis CCC inwhich thehigher stationary phase retention was obtained at 800 rpm

Nomenclature

1198772 Correlation index

1198772

=

(sum119899

119894=1119909119894119910119894minus sum119899

119894=1119909119894sum119899

119894=1119910119894119899)2

[sum119899

119894=1119909119894

2minus (sum119899

119894=1119909119894)2

119899] [sum119899

119894=1119910119894

2minus (sum119899

119894=1119910119894)2

119899]

(lowast)

Journal of Fluids 9

where 119909119894 119910119894 and 119899 are the experimental calculated

and number of data points respectivelyAARD Average absolute relative deviation

AARD = (

119899

sum

119894=1

100381610038161003816100381610038161003816100381610038161003816

119910exp119894 minus 119910cal119894

119910exp119894times 100

100381610038161003816100381610038161003816100381610038161003816

) times (

1

119899

) (lowastlowast)

where 119910exp119894 119910cal119894 and 119899 are the experimental calcu-lated and number of data points respectively

Δℎ Height of the interphaseΔ119905 Time of phase separation119881tp Volume of top phase119881bp Volume of bottom phase1198720 Morton number

Greek Letters

Δ120588 Density difference between the top andbottomphases120588119888 Density of the continuous phase

120574 Interfacial tension between the top and bottomphases120590119908 Surface tension of the air-water interface at 25∘C

120590119888 Surface tension between air-continuous phase inter-face

120583119888 Viscosity of the continuous phase

120583119889 Viscosity of the dispersed phase

120583119887 Viscosity of the bottom-continuous phase

Δ120583 Viscosity difference between the phases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work is supported by the National Natural ScienceFoundation of China (no 21476135) Outstanding YoungTeachers Training Program in Guangdong Higher Edu-cation Institutions (no Yq2013076) Science and Technol-ogy Planning Project of Guangdong Province China (no2012B060400006) and Education Department Projects ofGuangdong Province China (nos 2012KJCX0052 and 920-38030337)

References

[1] J Benavides and M Rito-Palomares ldquoPractical experiencesfrom the development of aqueous two-phase processes for therecovery of high value biological productsrdquo Journal of ChemicalTechnology and Biotechnology vol 83 no 2 pp 133ndash142 2008

[2] J Benavides O Aguilar B H Lapizco-Encinas and M Rito-Palomares ldquoExtraction and purification of bioproducts andnanoparticles using aqueous two-phase systems strategiesrdquoChemical Engineering and Technology vol 31 no 6 pp 838ndash845 2008

[3] D F C Silva A M Azevedo P Fernandes V Chu J P Condeand M R Aires-Barros ldquoDesign of a microfluidic platform formonoclonal antibody extraction using an aqueous two-phasesystemrdquo Journal of Chromatography A vol 1249 pp 1ndash7 2012

[4] S L Mistry A Kaul J C Merchuk and J A Asenjo ldquoMath-ematical modelling and computer simulation of aqueous two-phase continuous protein extractionrdquo Journal of Chromatogra-phy A vol 741 no 2 pp 151ndash163 1996

[5] P A Albertsson Partition of Cell Particles and MacromoleculesWiley-Interscience New York NY USA 1986

[6] B Y Zaslasvsky Aqueous Two-Phase Partitioning PhysicalChemistry and Bioanalytical Applications Marcel Dekker NewYork NY USA 1995

[7] MRito-Palomares ldquoPractical application of aqueous two-phasepartition to process development for the recovery of biologicalproductsrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 807 no 1 pp 3ndash11 2004

[8] E Huenupi A Gomez B A Andrews and J A Asenjo ldquoOpti-mization and design considerations of two-phase continuousprotein separationrdquo Journal of Chemical Technology amp Biotech-nology vol 74 no 3 pp 256ndash263 1999

[9] S Saravanan J R Rao T Murugesan B U Nair and TRamasami ldquoRecovery of value-added globular proteins fromtannery wastewaters using PEGmdashsalt aqueous two-phase sys-temsrdquo Journal of Chemical Technology amp Biotechnology vol 81no 11 pp 1814ndash1819 2006

[10] P A J Rosa I F Ferreira A M Azevedo and M R Aires-Barros ldquoAqueous two-phase systems a viable platform in themanufacturing of biopharmaceuticalsrdquo Journal of Chromatog-raphy A vol 1217 no 16 pp 2296ndash2305 2010

[11] K H Kroner H Schutte W Stach and M-R Kula ldquoScale-up of formate dehydrogenase by partitionrdquo Journal of ChemicalTechnology and Biotechnology vol 32 no 1 pp 130ndash137 1982

[12] K Selber F Tjerneld A Collen et al ldquoLarge-scale separationand production of engineered proteins designed for facilitatedrecovery in detergent-based aqueous two-phase extractionsystemsrdquo Process Biochemistry vol 39 no 7 pp 889ndash896 2004

[13] Y ZengG Liu YMaX YChen andY Ito ldquoOrganic high ionicstrength aqueous two-phase solvent system series for separationof ultra-polar compounds by spiral high-speed counter-currentchromatographyrdquo Journal of Chromatography A vol 1218 no48 pp 8715ndash8717 2011

[14] M H Salamanca J C Merchuk B A Andrews and J AAsenjo ldquoOn the kinetics of phase separation in aqueous two-phase systemsrdquo Journal of Chromatography B Biomedical Appli-cations vol 711 no 1-2 pp 319ndash329 1998

[15] A VNarayanMCMadhusudhan andK SM S RaghavaraoldquoDemixing kinetics of phase systems employed for liquid-liquidextraction and correlation with system propertiesrdquo Food andBioproducts Processing vol 89 no 4 pp 251ndash256 2011

[16] K Shinomiya H Kobayashi N Inokuchi et al ldquoNew small-scale cross-axis coil planet centrifuge Partition efficiency andapplication to purification of bullfrog ribonucleaserdquo Journal ofChromatography A vol 1151 no 1-2 pp 91ndash98 2007

[17] NMekaoui K Faure andA Berthod ldquoAdvances in countercur-rent chromatography for protein separationsrdquo Bioanalysis vol4 no 7 pp 833ndash844 2012

10 Journal of Fluids

[18] K Shinomiya H Kobayashi N Inokuchi K Nakagomi and YIto ldquoPartition efficiency of high-pitch locular multilayer coil forcountercurrent chromatographic separation of proteins usingsmall-scale cross-axis coil planet centrifuge and applicationto purification of various collagenases with aqueous-aqueouspolymer phase systemsrdquo Journal of Liquid Chromatography ampRelated Technologies vol 34 no 3 pp 182ndash194 2011

[19] G Tubio G A Pico and B B Nerli ldquoExtraction of trypsinfrom bovine pancreas by applying polyethyleneglycolsodiumcitrate aqueous two-phase systemsrdquo Journal of ChromatographyB Analytical Technologies in the Biomedical and Life Sciencesvol 877 no 3 pp 115ndash120 2009

[20] R L Perez D B Loureiro B B Nerli and G Tubio ldquoOptimiza-tion of pancreatic trypsin extraction in PEGcitrate aqueoustwo-phase systemsrdquo Protein Expression and Purificatio vol 106pp 66ndash71 2015

[21] M Perumalsamy and T Murugesan ldquoPrediction of liquid-liquid equilibria for PEG 2000-sodium citrate based aqueoustwo-phase systemsrdquo Fluid Phase Equilibria vol 244 no 1 pp52ndash61 2006

[22] Y Liu Y Q Feng and Y J Zhao ldquoLiquidndashliquid equilibriumof various aqueous two-phase systems experiment and corre-lationrdquo Journal of Chemical amp Engineering Data vol 58 no 10pp 2775ndash2784 2013

[23] M C Wikinson ldquoExtended use of and comments on thedrop-weight (drop-volume) technique for the determination ofsurface and interfacial tensionsrdquo Journal of Colloid and InterfaceScience vol 40 pp 14ndash26 1972

[24] C Jho and R Burke ldquoDrop weight technique for the mea-surement of dynamic surface tensionrdquo Journal of Colloid andInterface Science vol 95 no 1 pp 61ndash71 1983

[25] J A Asenjo S L Mistry B A Andrews and J C MerchukldquoPhase separation rates of aqueous two-phase systems correla-tion with system propertiesrdquo Biotechnology and Bioengineeringvol 79 no 2 pp 217ndash223 2002

[26] E Barnea and J Mizrahi ldquoSeparation mechanism of liquid-liquid dispersions in a deep-layer gravity settler part IV-continuous settler characteristicsrdquo Transactions of the Institu-tion of Chemical Engineers vol 56 pp 83ndash92 1975

[27] J Golob and R Modic ldquoCoalescence of liquidliquid disper-sions in gravity settlersrdquo Transactions of IChemE vol 55 pp207ndash211 1977

[28] A Kaul R A M Pereira J A Asenjo and J C MerchukldquoKinetics of phase separation for polyethylene glycol-phosphatetwo-phase systemsrdquo Biotechnology and Bioengineering vol 48no 3 pp 246ndash256 1995

[29] M T Zafarani-Moattar R Sadeghi and A A Hamidi ldquoLiquid-liquid equilibria of an aqueous two-phase system containingpolyethylene glycol and sodium citrate experiment and corre-lationrdquo Fluid Phase Equilibria vol 219 no 2 pp 149ndash155 2004

[30] V H Nagaraja and R Lyyaswami ldquoPhase demixing studies inaqueous two-phase system with polyethylene glycol (PEG) andsodium citraterdquo Chemical Engineering Communications vol200 no 10 pp 1293ndash1308 2013

[31] T Murugesan and I Regupathi ldquoPrediction of continuousphase axial mixing in rotating disc contactorsrdquo Journal ofChemical Engineering of Japan vol 37 no 10 pp 1293ndash13022004

[32] K Shinomiya K Yanagidaira and Y Ito ldquoNew small-scalecross-axis coil planet centrifuge the design of the apparatus andits application to counter-current chromatographic separation

of proteins with aqueous-aqueous polymer phase systemsrdquoJournal of Chromatography A vol 1104 no 1-2 pp 245ndash2552006

[33] Y Shibusawa Y Eriguchi and Y Ito ldquoPurification of lacticacid dehydrogenase from bovine heart crude extract bycounter-current chromatographyrdquo Journal of ChromatographyB Biomedical Applications vol 696 no 1 pp 25ndash31 1997

[34] M Perumalsamy and T Murugesan ldquoPhase compositionsmolarmass and temperature effect on densities viscosities andliquid-liquid equilibrium of polyethylene glycol and salt-basedaqueous two-phase systemsrdquo Journal of Chemical amp EngineeringData vol 54 no 4 pp 1359ndash1366 2009

[35] K Mishima K Matsuyama M Ezawa Y Taruta S Takarabeand M Nagatani ldquoInterfacial tension of aqueous two-phasesystems containing poly(ethylene glycol) and dipotassiumhydrogenphosphaterdquo Journal of Chromatography B BiomedicalApplications vol 711 no 1-2 pp 313ndash318 1998

[36] Y Ito ldquoGolden rules and pitfalls in selecting optimum condi-tions for high-speed counter-current chromatographyrdquo Journalof Chromatography A vol 1065 no 2 pp 145ndash168 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

High Energy PhysicsAdvances in

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


FluidsJournal of

Atomic and Molecular Physics

Journal of



Advances in Condensed Matter Physics

OpticsInternational Journal of



AstronomyAdvances in

International Journal of


Superconductivity


Statistical MechanicsInternational Journal of


GravityJournal of


AstrophysicsJournal of


Physics Research International


Solid State PhysicsJournal of

Computational Methods in Physics

Journal of



Soft MatterJournal of

Hindawi Publishing Corporationhttpwwwhindawicom

AerodynamicsJournal of

Volume 2014


PhotonicsJournal of


Journal of

Biophysics


ThermodynamicsJournal of

2 Journal of Fluids

Many works focused on HSCCC instrument design forATPS separation to eliminate the emulsion [16ndash18] Thereare few reports about the phase separation rate and physicalproperties of ATPS [14 15] for HSCCC

Much of the research [19 20] in ATPS has found thatpolyethylene glycolorganic salts ATPS could be employedas a viable and potentially useful tool for separating proteinsinstead of the conventional PEGinorganic systemsThemainadvantages of PEGsodium citrate ATPS are the biodegrad-ability and nontoxicity of the organic anion when comparingwith the high eutrophication potential of inorganic ions [21]and its harmful impact on the environment In our presentwork the physical properties and phase separation behav-iors of the ATPS including PEGorganic salts (PEGsodiumcitrate) and PEGinorganic salts (PEGammonium sulfate)systemswere investigated for comparisons Here ammoniumsulfate was selected as the inorganic salt due to its frequentuse in protein precipitation The phase separation rate andthe physical properties of ATPS such as density viscosity andinterfacial tension were measured and correlated with the tieline length (TLL) and ATPS 119881

119903which can contribute to the

HSCCC application using ATPS for protein separationFinally the stationary phase retention of PEGsodium citrateATPS in the cross-axis countercurrent chromatography (119883-axis CCC) was investigated based on the above correlatedmodels in which the 119883-axis CCC was designed and fabri-cated in our laboratory

2 Materials and Methods

21 Chemicals PEG1000 and PEG4000 (GR ge 95 masspurity) were purchased from Merck (Shanghai China)Sodium citrate (GR ge 995 mass purity) and ammoniumsulfate (GR ge 995 mass purity) were of analytical gradefrom local sources Aqueous solutions were prepared withdeionized and doubly distilled water

22 Preparation of ATPS ATPS were prepared from stocksolutions of PEG (50ww) sodium citrate (30ww) andammonium sulfate (40ww) Phase boundaries of all ATPSwere obtained by cloud-point measurements in a thermo-static bath (DC-0506 Shanghai Jingtian Electronic Instru-ment Co Ltd) of 25∘CThe temperature was maintained withan uncertainty of plusmn005∘C The PEGsalt ATPS with five tielines lengths (30 35 40 45 and 50) were equilibrated for 12 hat 25 plusmn 005

∘C before use The compositions of the tie lineswere determined by the119881

119903and the lever rule [22]The119881

119903was

determined in graduated centrifuge tubeswith an uncertaintyof plusmn01mL

23 Density Measurements The top and bottom phase den-sities were measured using a 10mL density bottle with anuncertainty of plusmn01 kgsdotmminus3 at 25 plusmn 005

∘CThemeasurementswere done in triplicate and the average value was reported

24 Viscosity Measurements The viscosity of all ATPS wasmeasured using digital rotary viscosimeter (NDJ-5S Shang-hai Jingtian Electronic Instrument Co Ltd) with a precision

of plusmn00001 PasdotsThe samples were first maintained at workingtemperature in thermostatic bath with an uncertainty ofplusmn005∘C for 10min and measurements were done in tripli-cate

25 Interfacial TensionMeasurements The interfacial tensionwas measured by a drop volume method [23 24] at 25∘CSome burettes having capillaries with different outer diame-ters were used since the interfacial tension changed with thecomposition of ATPS Interfacial tension between the twophases was determined by the numbers and volumes of fallendrops from the top phase solution and the density differencebetween the top and bottom phasesThe interfacial tension isthen given by

120574 =

Δ120588119881119892

2120587119903120595

120595 = 09045 minus 07294 (

119903

11988113

) + 04293 (

119903

11988113

)

2

(03 lt

119903

11988113

lt 12)

120595 = 1007 minus 1479 (

119903

11988113

) + 1829 (

119903

11988113

)

2

(

119903

11988113

le 03)

(1)

where 120574 is the interfacial tension between the phases of ATPSΔ120588 is the density difference of the two phases119881 is the averagevolume of drops 119903 is the radius of the tip of burette 119892 isthe local value for the acceleration due to gravity and 120595 isthe correction factors determined by the values of 119881 and 119903The uncertainty of interfacial tension measurement wasplusmn00001mNsdotmminus1 All the measurements were done in trip-licate

26 Phase Separation Rate ATPS mixing was performed incentrifuge tubes with 10mL volume The time of mixing was10min bymanual operation For all the experiments the timefor phase separation was defined as the time needed for mostbulk of the phases to separate and a horizontal interface wasformed Some small drops of one phase which remained inthe other phase were ignored The measurements were donein triplicate with a precision of plusmn01 s

Kaul et al found that the kinetic behavior depends greatlyon which of the phases is continuous and that the propertiesof the continuous phase strongly influence the movement ofthe drops of the dispersed phase [25ndash27] Two typical batchesrsquoseparation is shown in Figure 1 For the top-continuousATPSthe drops of bottom phase descend in centrifuge tubes andform a settling front for the bottom-continuous ATPS thedrops of top phase ascend in centrifuge tubes and form arising front It can be seen that the process of coalescence isslower than droplet descent and ascent due to the existence ofthe front

The problems of phase continuity and phase inversionare important for the phase separation behavior and phase

Journal of Fluids 3







119903was investi-





0) the ratio


119889ℎ

119889119905

(

120583119888

120582

) = 1198861198720

119887

(

120574

120590119888

)

119888

(

119881tp

119881bp)

119889

(TLL)119890 (2)

1198720=

Δ1205834

119892

1205743Δ120588

(3)











4 Journal of Fluids

(a) (b)


25 30 35 40 45 50 55

108

112

116

120

124

Den

sity

(gm

L)

TLL (ww)

(a)

114

117

111

108

105

Den

sity

(gm

L)

25 30 35 40 45 50 55

TLL (ww)

(b)

108

112

116

120

124

128

Den

sity

(gm

L)

25 30 35 40 45 50 55

TLL (ww)

TopBottom

(c)

108

104

112

116

120

124

Den

sity

(gm

L)

25 30 35 40 45 50 55

TLL (ww)

TopBottom

(d)


Journal of Fluids 5

TLL (ww)28 32 36 40 44 48 52

0

10

20

30

40120578middot103

(Pamiddot

s)

(a)

0

80

20

40

60

120578middot103

(Pamiddot

s)

TLL (ww)28 32 36 40 44 48 52

(b)

TLL (ww)28 32 36 40 44 48 52

0

6

18

12

24

120578middot103

(Pamiddot

s)

TopBottom

(c)

TLL (ww)28 32 36 40 44 48 52

TopBottom

0

80

100

20

40

60120578middot103

(Pamiddot

s)

(d)



119903= 1 were



119903+ 1))


119903=



119905is











119889ℎ

119889119905

(

120583119888

120582

) = 1198861198720

119887

(

120574

120590119908

)

119888

(TLL)119889 (4)

6 Journal of Fluids

TLL (ww)28 34 40 46 52

060

080

100

120

140

Inte

rfaci

al te

nsio

n (m

Nm

)

(a)

TLL (ww)28 34 40 46 52

000

020

040

060

080

100

Inte

rfaci

al te

nsio

n (m

Nm

)

(b)

TLL (ww)28 34 40 46 52

000

010

020

030

040

050

Inte

rfaci

al te

nsio

n (m

Nm

)

(c)

TLL (ww)28 34 40 46 52

000

020

040

060

080

Inte

rfaci

al te

nsio

n (m

Nm

)

(d)




119889ℎ

119889119905

(

120583119887

120574


1198720

00080

(

120574

120590119908

)

minus18688

(TLL)07650

(5)


119889ℎ

119889119905

(

120583119887

120574


1198720

01735

(

120574

120590119908

)

minus15403

(TLL)25750

(6)













119903dependence

Journal of Fluids 7

0

0

01

01

02

02

03

03

04

04

05


PEG1000

(w

w)

(a)

0

01

02

03

04

05

06

0 01 02 03


PEG4000

(w

w)

(b)

0 01 02 03 04

Sodium citrate (ww)

0

01

02

03

04

05

PEG1000

(w

w)

(c)

0 01 02 03

Sodium citrate (ww)

000

010

020

030

040

050

PEG4000

(w

w)

(d)


000 020 040 060 080 100

0

200

400

600

800


Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(a)

0

100

200

300

400

500

000 020 040 060 080 100


Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(b)


= 1(119881119903


8 Journal of Fluids


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 9038 7197 0739 22130 212035 9346 7197 0846 24158 219340 9752 7197 0965 27053 214845 9644 7197 1140 28595 220550 10104 7197 1161 32305 2295


30 28062 7197 0272 25010 199035 24702 7197 0376 33085 186540 21888 7197 0439 41625 177545 18800 7197 0592 54005 189550 16410 7197 0805 67790 2210


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 15618 7197 0083 5650 365035 13348 7197 0169 7780 372040 12202 7197 0249 9500 405045 11198 7197 0297 1168 442050 11132 7197 0405 14365 5235


30 27993 7197 0167 23165 243535 24785 7197 0305 31185 271540 24429 7197 0433 42110 294045 26705 7197 0579 55145 335550 26648 7197 0755 71985 4015


3873

4438

4923

54835144

5041

30

35

40

45

50

55

60

400 500 600 700 800 900 1000 1100

Stat

iona

ry p

hase

rete

ntio

n (

)



4 Conclusion




119903value and the





119903= 1 The


Nomenclature


1198772

=

(sum119899

119894=1119909119894119910119894minus sum119899

119894=1119909119894sum119899

119894=1119910119894119899)2

[sum119899

119894=1119909119894

2minus (sum119899

119894=1119909119894)2

119899] [sum119899

119894=1119910119894

2minus (sum119899

119894=1119910119894)2

119899]

(lowast)

Journal of Fluids 9



AARD = (

119899

sum

119894=1

100381610038161003816100381610038161003816100381610038161003816

119910exp119894 minus 119910cal119894

119910exp119894times 100

100381610038161003816100381610038161003816100381610038161003816

) times (

1

119899

) (lowastlowast)



Greek Letters










Acknowledgments


References












































FluidsJournal of


Journal of










Superconductivity




GravityJournal of








Journal of






Volume 2014


PhotonicsJournal of


Journal of

Biophysics



Journal of Fluids 3







119903was investi-





0) the ratio


119889ℎ

119889119905

(

120583119888

120582

) = 1198861198720

119887

(

120574

120590119888

)

119888

(

119881tp

119881bp)

119889

(TLL)119890 (2)

1198720=

Δ1205834

119892

1205743Δ120588

(3)











4 Journal of Fluids

(a) (b)


25 30 35 40 45 50 55

108

112

116

120

124

Den

sity

(gm

L)

TLL (ww)

(a)

114

117

111

108

105

Den

sity

(gm

L)

25 30 35 40 45 50 55

TLL (ww)

(b)

108

112

116

120

124

128

Den

sity

(gm

L)

25 30 35 40 45 50 55

TLL (ww)

TopBottom

(c)

108

104

112

116

120

124

Den

sity

(gm

L)

25 30 35 40 45 50 55

TLL (ww)

TopBottom

(d)


Journal of Fluids 5

TLL (ww)28 32 36 40 44 48 52

0

10

20

30

40120578middot103

(Pamiddot

s)

(a)

0

80

20

40

60

120578middot103

(Pamiddot

s)

TLL (ww)28 32 36 40 44 48 52

(b)

TLL (ww)28 32 36 40 44 48 52

0

6

18

12

24

120578middot103

(Pamiddot

s)

TopBottom

(c)

TLL (ww)28 32 36 40 44 48 52

TopBottom

0

80

100

20

40

60120578middot103

(Pamiddot

s)

(d)



119903= 1 were



119903+ 1))


119903=



119905is











119889ℎ

119889119905

(

120583119888

120582

) = 1198861198720

119887

(

120574

120590119908

)

119888

(TLL)119889 (4)

6 Journal of Fluids

TLL (ww)28 34 40 46 52

060

080

100

120

140

Inte

rfaci

al te

nsio

n (m

Nm

)

(a)

TLL (ww)28 34 40 46 52

000

020

040

060

080

100

Inte

rfaci

al te

nsio

n (m

Nm

)

(b)

TLL (ww)28 34 40 46 52

000

010

020

030

040

050

Inte

rfaci

al te

nsio

n (m

Nm

)

(c)

TLL (ww)28 34 40 46 52

000

020

040

060

080

Inte

rfaci

al te

nsio

n (m

Nm

)

(d)




119889ℎ

119889119905

(

120583119887

120574


1198720

00080

(

120574

120590119908

)

minus18688

(TLL)07650

(5)


119889ℎ

119889119905

(

120583119887

120574


1198720

01735

(

120574

120590119908

)

minus15403

(TLL)25750

(6)













119903dependence

Journal of Fluids 7

0

0

01

01

02

02

03

03

04

04

05


PEG1000

(w

w)

(a)

0

01

02

03

04

05

06

0 01 02 03


PEG4000

(w

w)

(b)

0 01 02 03 04

Sodium citrate (ww)

0

01

02

03

04

05

PEG1000

(w

w)

(c)

0 01 02 03

Sodium citrate (ww)

000

010

020

030

040

050

PEG4000

(w

w)

(d)


000 020 040 060 080 100

0

200

400

600

800


Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(a)

0

100

200

300

400

500

000 020 040 060 080 100


Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(b)


= 1(119881119903


8 Journal of Fluids


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 9038 7197 0739 22130 212035 9346 7197 0846 24158 219340 9752 7197 0965 27053 214845 9644 7197 1140 28595 220550 10104 7197 1161 32305 2295


30 28062 7197 0272 25010 199035 24702 7197 0376 33085 186540 21888 7197 0439 41625 177545 18800 7197 0592 54005 189550 16410 7197 0805 67790 2210


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 15618 7197 0083 5650 365035 13348 7197 0169 7780 372040 12202 7197 0249 9500 405045 11198 7197 0297 1168 442050 11132 7197 0405 14365 5235


30 27993 7197 0167 23165 243535 24785 7197 0305 31185 271540 24429 7197 0433 42110 294045 26705 7197 0579 55145 335550 26648 7197 0755 71985 4015


3873

4438

4923

54835144

5041

30

35

40

45

50

55

60

400 500 600 700 800 900 1000 1100

Stat

iona

ry p

hase

rete

ntio

n (

)



4 Conclusion




119903value and the





119903= 1 The


Nomenclature


1198772

=

(sum119899

119894=1119909119894119910119894minus sum119899

119894=1119909119894sum119899

119894=1119910119894119899)2

[sum119899

119894=1119909119894

2minus (sum119899

119894=1119909119894)2

119899] [sum119899

119894=1119910119894

2minus (sum119899

119894=1119910119894)2

119899]

(lowast)

Journal of Fluids 9



AARD = (

119899

sum

119894=1

100381610038161003816100381610038161003816100381610038161003816

119910exp119894 minus 119910cal119894

119910exp119894times 100

100381610038161003816100381610038161003816100381610038161003816

) times (

1

119899

) (lowastlowast)



Greek Letters










Acknowledgments


References












































FluidsJournal of


Journal of










Superconductivity




GravityJournal of








Journal of






Volume 2014


PhotonicsJournal of


Journal of

Biophysics



4 Journal of Fluids

(a) (b)


25 30 35 40 45 50 55

108

112

116

120

124

Den

sity

(gm

L)

TLL (ww)

(a)

114

117

111

108

105

Den

sity

(gm

L)

25 30 35 40 45 50 55

TLL (ww)

(b)

108

112

116

120

124

128

Den

sity

(gm

L)

25 30 35 40 45 50 55

TLL (ww)

TopBottom

(c)

108

104

112

116

120

124

Den

sity

(gm

L)

25 30 35 40 45 50 55

TLL (ww)

TopBottom

(d)


Journal of Fluids 5

TLL (ww)28 32 36 40 44 48 52

0

10

20

30

40120578middot103

(Pamiddot

s)

(a)

0

80

20

40

60

120578middot103

(Pamiddot

s)

TLL (ww)28 32 36 40 44 48 52

(b)

TLL (ww)28 32 36 40 44 48 52

0

6

18

12

24

120578middot103

(Pamiddot

s)

TopBottom

(c)

TLL (ww)28 32 36 40 44 48 52

TopBottom

0

80

100

20

40

60120578middot103

(Pamiddot

s)

(d)



119903= 1 were



119903+ 1))


119903=



119905is











119889ℎ

119889119905

(

120583119888

120582

) = 1198861198720

119887

(

120574

120590119908

)

119888

(TLL)119889 (4)

6 Journal of Fluids

TLL (ww)28 34 40 46 52

060

080

100

120

140

Inte

rfaci

al te

nsio

n (m

Nm

)

(a)

TLL (ww)28 34 40 46 52

000

020

040

060

080

100

Inte

rfaci

al te

nsio

n (m

Nm

)

(b)

TLL (ww)28 34 40 46 52

000

010

020

030

040

050

Inte

rfaci

al te

nsio

n (m

Nm

)

(c)

TLL (ww)28 34 40 46 52

000

020

040

060

080

Inte

rfaci

al te

nsio

n (m

Nm

)

(d)




119889ℎ

119889119905

(

120583119887

120574


1198720

00080

(

120574

120590119908

)

minus18688

(TLL)07650

(5)


119889ℎ

119889119905

(

120583119887

120574


1198720

01735

(

120574

120590119908

)

minus15403

(TLL)25750

(6)













119903dependence

Journal of Fluids 7

0

0

01

01

02

02

03

03

04

04

05


PEG1000

(w

w)

(a)

0

01

02

03

04

05

06

0 01 02 03


PEG4000

(w

w)

(b)

0 01 02 03 04

Sodium citrate (ww)

0

01

02

03

04

05

PEG1000

(w

w)

(c)

0 01 02 03

Sodium citrate (ww)

000

010

020

030

040

050

PEG4000

(w

w)

(d)


000 020 040 060 080 100

0

200

400

600

800


Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(a)

0

100

200

300

400

500

000 020 040 060 080 100


Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(b)


= 1(119881119903


8 Journal of Fluids


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 9038 7197 0739 22130 212035 9346 7197 0846 24158 219340 9752 7197 0965 27053 214845 9644 7197 1140 28595 220550 10104 7197 1161 32305 2295


30 28062 7197 0272 25010 199035 24702 7197 0376 33085 186540 21888 7197 0439 41625 177545 18800 7197 0592 54005 189550 16410 7197 0805 67790 2210


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 15618 7197 0083 5650 365035 13348 7197 0169 7780 372040 12202 7197 0249 9500 405045 11198 7197 0297 1168 442050 11132 7197 0405 14365 5235


30 27993 7197 0167 23165 243535 24785 7197 0305 31185 271540 24429 7197 0433 42110 294045 26705 7197 0579 55145 335550 26648 7197 0755 71985 4015


3873

4438

4923

54835144

5041

30

35

40

45

50

55

60

400 500 600 700 800 900 1000 1100

Stat

iona

ry p

hase

rete

ntio

n (

)



4 Conclusion




119903value and the





119903= 1 The


Nomenclature


1198772

=

(sum119899

119894=1119909119894119910119894minus sum119899

119894=1119909119894sum119899

119894=1119910119894119899)2

[sum119899

119894=1119909119894

2minus (sum119899

119894=1119909119894)2

119899] [sum119899

119894=1119910119894

2minus (sum119899

119894=1119910119894)2

119899]

(lowast)

Journal of Fluids 9



AARD = (

119899

sum

119894=1

100381610038161003816100381610038161003816100381610038161003816

119910exp119894 minus 119910cal119894

119910exp119894times 100

100381610038161003816100381610038161003816100381610038161003816

) times (

1

119899

) (lowastlowast)



Greek Letters










Acknowledgments


References












































FluidsJournal of


Journal of










Superconductivity




GravityJournal of








Journal of






Volume 2014


PhotonicsJournal of


Journal of

Biophysics



Journal of Fluids 5

TLL (ww)28 32 36 40 44 48 52

0

10

20

30

40120578middot103

(Pamiddot

s)

(a)

0

80

20

40

60

120578middot103

(Pamiddot

s)

TLL (ww)28 32 36 40 44 48 52

(b)

TLL (ww)28 32 36 40 44 48 52

0

6

18

12

24

120578middot103

(Pamiddot

s)

TopBottom

(c)

TLL (ww)28 32 36 40 44 48 52

TopBottom

0

80

100

20

40

60120578middot103

(Pamiddot

s)

(d)



119903= 1 were



119903+ 1))


119903=



119905is











119889ℎ

119889119905

(

120583119888

120582

) = 1198861198720

119887

(

120574

120590119908

)

119888

(TLL)119889 (4)

6 Journal of Fluids

TLL (ww)28 34 40 46 52

060

080

100

120

140

Inte

rfaci

al te

nsio

n (m

Nm

)

(a)

TLL (ww)28 34 40 46 52

000

020

040

060

080

100

Inte

rfaci

al te

nsio

n (m

Nm

)

(b)

TLL (ww)28 34 40 46 52

000

010

020

030

040

050

Inte

rfaci

al te

nsio

n (m

Nm

)

(c)

TLL (ww)28 34 40 46 52

000

020

040

060

080

Inte

rfaci

al te

nsio

n (m

Nm

)

(d)




119889ℎ

119889119905

(

120583119887

120574


1198720

00080

(

120574

120590119908

)

minus18688

(TLL)07650

(5)


119889ℎ

119889119905

(

120583119887

120574


1198720

01735

(

120574

120590119908

)

minus15403

(TLL)25750

(6)













119903dependence

Journal of Fluids 7

0

0

01

01

02

02

03

03

04

04

05


PEG1000

(w

w)

(a)

0

01

02

03

04

05

06

0 01 02 03


PEG4000

(w

w)

(b)

0 01 02 03 04

Sodium citrate (ww)

0

01

02

03

04

05

PEG1000

(w

w)

(c)

0 01 02 03

Sodium citrate (ww)

000

010

020

030

040

050

PEG4000

(w

w)

(d)


000 020 040 060 080 100

0

200

400

600

800


Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(a)

0

100

200

300

400

500

000 020 040 060 080 100


Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(b)


= 1(119881119903


8 Journal of Fluids


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 9038 7197 0739 22130 212035 9346 7197 0846 24158 219340 9752 7197 0965 27053 214845 9644 7197 1140 28595 220550 10104 7197 1161 32305 2295


30 28062 7197 0272 25010 199035 24702 7197 0376 33085 186540 21888 7197 0439 41625 177545 18800 7197 0592 54005 189550 16410 7197 0805 67790 2210


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 15618 7197 0083 5650 365035 13348 7197 0169 7780 372040 12202 7197 0249 9500 405045 11198 7197 0297 1168 442050 11132 7197 0405 14365 5235


30 27993 7197 0167 23165 243535 24785 7197 0305 31185 271540 24429 7197 0433 42110 294045 26705 7197 0579 55145 335550 26648 7197 0755 71985 4015


3873

4438

4923

54835144

5041

30

35

40

45

50

55

60

400 500 600 700 800 900 1000 1100

Stat

iona

ry p

hase

rete

ntio

n (

)



4 Conclusion




119903value and the





119903= 1 The


Nomenclature


1198772

=

(sum119899

119894=1119909119894119910119894minus sum119899

119894=1119909119894sum119899

119894=1119910119894119899)2

[sum119899

119894=1119909119894

2minus (sum119899

119894=1119909119894)2

119899] [sum119899

119894=1119910119894

2minus (sum119899

119894=1119910119894)2

119899]

(lowast)

Journal of Fluids 9



AARD = (

119899

sum

119894=1

100381610038161003816100381610038161003816100381610038161003816

119910exp119894 minus 119910cal119894

119910exp119894times 100

100381610038161003816100381610038161003816100381610038161003816

) times (

1

119899

) (lowastlowast)



Greek Letters










Acknowledgments


References












































FluidsJournal of


Journal of










Superconductivity




GravityJournal of








Journal of






Volume 2014


PhotonicsJournal of


Journal of

Biophysics



6 Journal of Fluids

TLL (ww)28 34 40 46 52

060

080

100

120

140

Inte

rfaci

al te

nsio

n (m

Nm

)

(a)

TLL (ww)28 34 40 46 52

000

020

040

060

080

100

Inte

rfaci

al te

nsio

n (m

Nm

)

(b)

TLL (ww)28 34 40 46 52

000

010

020

030

040

050

Inte

rfaci

al te

nsio

n (m

Nm

)

(c)

TLL (ww)28 34 40 46 52

000

020

040

060

080

Inte

rfaci

al te

nsio

n (m

Nm

)

(d)




119889ℎ

119889119905

(

120583119887

120574


1198720

00080

(

120574

120590119908

)

minus18688

(TLL)07650

(5)


119889ℎ

119889119905

(

120583119887

120574


1198720

01735

(

120574

120590119908

)

minus15403

(TLL)25750

(6)













119903dependence

Journal of Fluids 7

0

0

01

01

02

02

03

03

04

04

05


PEG1000

(w

w)

(a)

0

01

02

03

04

05

06

0 01 02 03


PEG4000

(w

w)

(b)

0 01 02 03 04

Sodium citrate (ww)

0

01

02

03

04

05

PEG1000

(w

w)

(c)

0 01 02 03

Sodium citrate (ww)

000

010

020

030

040

050

PEG4000

(w

w)

(d)


000 020 040 060 080 100

0

200

400

600

800


Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(a)

0

100

200

300

400

500

000 020 040 060 080 100


Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(b)


= 1(119881119903


8 Journal of Fluids


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 9038 7197 0739 22130 212035 9346 7197 0846 24158 219340 9752 7197 0965 27053 214845 9644 7197 1140 28595 220550 10104 7197 1161 32305 2295


30 28062 7197 0272 25010 199035 24702 7197 0376 33085 186540 21888 7197 0439 41625 177545 18800 7197 0592 54005 189550 16410 7197 0805 67790 2210


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 15618 7197 0083 5650 365035 13348 7197 0169 7780 372040 12202 7197 0249 9500 405045 11198 7197 0297 1168 442050 11132 7197 0405 14365 5235


30 27993 7197 0167 23165 243535 24785 7197 0305 31185 271540 24429 7197 0433 42110 294045 26705 7197 0579 55145 335550 26648 7197 0755 71985 4015


3873

4438

4923

54835144

5041

30

35

40

45

50

55

60

400 500 600 700 800 900 1000 1100

Stat

iona

ry p

hase

rete

ntio

n (

)



4 Conclusion




119903value and the





119903= 1 The


Nomenclature


1198772

=

(sum119899

119894=1119909119894119910119894minus sum119899

119894=1119909119894sum119899

119894=1119910119894119899)2

[sum119899

119894=1119909119894

2minus (sum119899

119894=1119909119894)2

119899] [sum119899

119894=1119910119894

2minus (sum119899

119894=1119910119894)2

119899]

(lowast)

Journal of Fluids 9



AARD = (

119899

sum

119894=1

100381610038161003816100381610038161003816100381610038161003816

119910exp119894 minus 119910cal119894

119910exp119894times 100

100381610038161003816100381610038161003816100381610038161003816

) times (

1

119899

) (lowastlowast)



Greek Letters










Acknowledgments


References












































FluidsJournal of


Journal of










Superconductivity




GravityJournal of








Journal of






Volume 2014


PhotonicsJournal of


Journal of

Biophysics



Journal of Fluids 7

0

0

01

01

02

02

03

03

04

04

05


PEG1000

(w

w)

(a)

0

01

02

03

04

05

06

0 01 02 03


PEG4000

(w

w)

(b)

0 01 02 03 04

Sodium citrate (ww)

0

01

02

03

04

05

PEG1000

(w

w)

(c)

0 01 02 03

Sodium citrate (ww)

000

010

020

030

040

050

PEG4000

(w

w)

(d)


000 020 040 060 080 100

0

200

400

600

800


Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(a)

0

100

200

300

400

500

000 020 040 060 080 100


Sepa

ratio

n tim

e (s)

TLL = 50

TLL = 40

(b)


= 1(119881119903


8 Journal of Fluids


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 9038 7197 0739 22130 212035 9346 7197 0846 24158 219340 9752 7197 0965 27053 214845 9644 7197 1140 28595 220550 10104 7197 1161 32305 2295


30 28062 7197 0272 25010 199035 24702 7197 0376 33085 186540 21888 7197 0439 41625 177545 18800 7197 0592 54005 189550 16410 7197 0805 67790 2210


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 15618 7197 0083 5650 365035 13348 7197 0169 7780 372040 12202 7197 0249 9500 405045 11198 7197 0297 1168 442050 11132 7197 0405 14365 5235


30 27993 7197 0167 23165 243535 24785 7197 0305 31185 271540 24429 7197 0433 42110 294045 26705 7197 0579 55145 335550 26648 7197 0755 71985 4015


3873

4438

4923

54835144

5041

30

35

40

45

50

55

60

400 500 600 700 800 900 1000 1100

Stat

iona

ry p

hase

rete

ntio

n (

)



4 Conclusion




119903value and the





119903= 1 The


Nomenclature


1198772

=

(sum119899

119894=1119909119894119910119894minus sum119899

119894=1119909119894sum119899

119894=1119910119894119899)2

[sum119899

119894=1119909119894

2minus (sum119899

119894=1119909119894)2

119899] [sum119899

119894=1119910119894

2minus (sum119899

119894=1119910119894)2

119899]

(lowast)

Journal of Fluids 9



AARD = (

119899

sum

119894=1

100381610038161003816100381610038161003816100381610038161003816

119910exp119894 minus 119910cal119894

119910exp119894times 100

100381610038161003816100381610038161003816100381610038161003816

) times (

1

119899

) (lowastlowast)



Greek Letters










Acknowledgments


References












































FluidsJournal of


Journal of










Superconductivity




GravityJournal of








Journal of






Volume 2014


PhotonicsJournal of


Journal of

Biophysics



8 Journal of Fluids


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 9038 7197 0739 22130 212035 9346 7197 0846 24158 219340 9752 7197 0965 27053 214845 9644 7197 1140 28595 220550 10104 7197 1161 32305 2295


30 28062 7197 0272 25010 199035 24702 7197 0376 33085 186540 21888 7197 0439 41625 177545 18800 7197 0592 54005 189550 16410 7197 0805 67790 2210


TLL Δ119905 120590119908

120574 Δ120583 120583119887



30 15618 7197 0083 5650 365035 13348 7197 0169 7780 372040 12202 7197 0249 9500 405045 11198 7197 0297 1168 442050 11132 7197 0405 14365 5235


30 27993 7197 0167 23165 243535 24785 7197 0305 31185 271540 24429 7197 0433 42110 294045 26705 7197 0579 55145 335550 26648 7197 0755 71985 4015


3873

4438

4923

54835144

5041

30

35

40

45

50

55

60

400 500 600 700 800 900 1000 1100

Stat

iona

ry p

hase

rete

ntio

n (

)



4 Conclusion




119903value and the





119903= 1 The


Nomenclature


1198772

=

(sum119899

119894=1119909119894119910119894minus sum119899

119894=1119909119894sum119899

119894=1119910119894119899)2

[sum119899

119894=1119909119894

2minus (sum119899

119894=1119909119894)2

119899] [sum119899

119894=1119910119894

2minus (sum119899

119894=1119910119894)2

119899]

(lowast)

Journal of Fluids 9



AARD = (

119899

sum

119894=1

100381610038161003816100381610038161003816100381610038161003816

119910exp119894 minus 119910cal119894

119910exp119894times 100

100381610038161003816100381610038161003816100381610038161003816

) times (

1

119899

) (lowastlowast)



Greek Letters










Acknowledgments


References












































FluidsJournal of


Journal of










Superconductivity




GravityJournal of








Journal of






Volume 2014


PhotonicsJournal of


Journal of

Biophysics



Journal of Fluids 9



AARD = (

119899

sum

119894=1

100381610038161003816100381610038161003816100381610038161003816

119910exp119894 minus 119910cal119894

119910exp119894times 100

100381610038161003816100381610038161003816100381610038161003816

) times (

1

119899

) (lowastlowast)



Greek Letters










Acknowledgments


References












































FluidsJournal of


Journal of










Superconductivity




GravityJournal of








Journal of






Volume 2014


PhotonicsJournal of


Journal of

Biophysics





























FluidsJournal of


Journal of










Superconductivity




GravityJournal of








Journal of






Volume 2014


PhotonicsJournal of


Journal of

Biophysics



research article phase separation behavior and system...

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